TWIK-related spinal cord K+ (TRESK) channel is abundantly expressed in all primary afferent neurons (PANs) in trigeminal ganglion (TG) and dorsal root ganglion (DRG), mediating background K+ currents and controlling the excitability of PANs. However, TRESK mutations cause migraine headache but not body pain in humans, suggesting that TG neurons are more vulnerable to TRESK dysfunctions than DRG neurons. We have found that the migraine-associated TRESK mutation results in the truncation of TRESK protein, which exerts a strong dominant-negative effect on endogenous TRESK current. This has prompted us to use TRESK knockout (KO) mouse as a model system to study the effects of de novo loss of TRESK activity on pain transmission. Despite the ubiquitous loss of TRESK currents in all PANs, only one subpopulation TG neurons in TRESK KO mice becomes hyper-excitable. Unexpectedly, the percentage of capsaicin-responsive neurons is selectively increased in TG but not DRG of KO mice. TRESK KO mice exhibit more robust behavioral responses than wild-type controls in mouse models of trigeminal pain, especially headache. In contrast, wild- type and KO mice respond similarly to noxious stimuli on the hindpaw. These results indicate that TRESK KO mice recapitulate the pathophysiology of TRESK mutations in humans. Although we have elucidated the mechanisms through which de novo TRESK dysfunction causes migraine in humans, we still don't know why ubiquitous loss of TRESK differentially affects TG and DRG neurons; whether changes of TRESK activity contribute to episodic and chronic migraines in general populations. In this project we propose to employ a multidisciplinary approach to address these knowledge gaps. First, we will investigate the mechanisms through which TRESK dysfunction differentially affects TG and DRG neurons. We will identify transcriptional regulatory proteins that selectively upregulate TRPV1 expression in KO TG neurons. We will also identify the ion channel(s) that compensate for the loss of TRESK activity in DRG neurons. Secondly, based on our preliminary finding that changes of endogenous TRESK activity correlates with changes of the excitability of TG neurons during estrous cycles in female mice, we will test the hypothesis that estrogen increases migraine susceptibility in women through inhibition of endogenous TRESK activity in TG neurons. Finally, we will test the hypothesis that frequent migraine attacks reduce TG TRESK currents, thereby increasing the risk of migraine chronification. We will investigate how endogenous TRESK activity is affected in a mouse model of chronic migraine; and whether altering TRESK activity in TG neurons is sufficient to modify the process of migraine chronification. Collectively, results from these studies will elucidate novel mechanisms through which TRESK differentially regulates the neuronal circuits encoding trigeminal and body pain. This will ultimately leads to better understanding of the physiology and pathophysiology of both systems.